Survival of cancer patients depends primarily on preventing the emergence of new tumors due to metastasis and requires better understanding of factors that allow the engraftment of circulating cancer cells in remote locations. In advanced tumors, diverse populations of immune cells interact with each other, the tumor, and the stroma to create a distinct microenvironment that can favor tumor growth or facilitate rejection. The good prognostic is infiltration by cytotoxic lymphocytes, primarily the innate NK cells, some gammadelta T cells, and tumor-specific CD8 effector T cells. In contrast, tumor infiltrating FoxP3 CD4 regulatory T cells, tumor associated macrophages, and certain dendritic cell populations correlate with aggressive tumor growth both in patients and experimental animals. Yet, how the balance between these and other immune cell populations evolves locally upon single metastatic cancer cell arrival in the healthy tissue remains unclear and needs to be studied in situ. Our goal is better understanding of the behavior of immune cells during early phases of tumor growth, which could suggest new ways to curb metastasis development. Our strategy is based on live imaging-based experimental framework that allows us to study the dynamics of endogenous populations of lymphocytes directly in the sites of nascent lung tumors beginning with single cancer cell arrival in the healthy tissue. Based on our preliminary studies, we hypothesize that the development of immunological microenvironment begins very early and involves a balancing act of recruitment, retention, and local activation of immuno-suppressory versus pro-inflammatory cells, triggered and modulated by cancer cell interaction with tissue- resident dendritic cells and macrophages. To understand the causative relationships, we propose to characterize in detail the dynamics of lymphocyte interactions during the earliest phases of metastasis establishment and to identify molecular determinants that are involved. This project will aid monitoring of new treatments directed at the immunological microenvironment of nascent tumors and may suggest new strategies to fight metastases formation. PUBLIC HEALTH RELEVANCE: Preventing the emergence of new tumors due to metastasis requires better understanding of factors that allow the engaftment of circulating cancer cells in healthy tissues. Using multiphoton intravital imaging of nascent lung metastases, we will characterize the recruitment and interactions of immune cells that can facilitate tumor growth or induce rejection. This study will help identify and target the earliest cell interactions for future antimetastatic therapies.